Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5′-end or 3′-end of the sense siRNA strand and the 5′-end of the antisense siRNA strand, but conjugation to the 3′-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2′-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5′-end of the guide strand coincided with the 5′-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5′-end of the sense strand, only 21-29 bp hairpins were highly active.